Carbonic acid, C,C'-[4-(2-bromoacetyl)-1,3-phenylene] C,C'-dimethyl ester

Endpoint summary

Description of key information

2’,4’-Bis(methoxycarbonyloxy)-2-brom-acetophenone was not irritating in an in vitro skin irritation test (reconstructed human epidermis test method according to OECD 439 and EU-Method B.46), as well as in an in vitro eye irritation test (BCOP Test according to OECD 437).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09.01.2015 - 19.01.2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Vehicle:

water

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Duration of post-treatment incubation (if applicable):

15 min

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Main experiment

Value:

> 93.2 - < 101.6

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

1. Pre-Experiments

The mixture of 10 mg test item per 2 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple. The mixture of 10 mg of the test item per 90 μL aqua dest. showed no colouring detectable by unaided eye-assessment.

2. Experiment

Table 1: Result of the test with 2’,4’-Bis(methoxycarbonyloxy)-2-brom-acetophenone

Name	Negative Control (NC)			Positive Control (PC)			Test Item
Tissue	1	2	3	1	2	3	1	2	3
absolute OD550	0.937 0.911	0.923 0.960	0.934 0.937	0.116 0.119	0.129 0.125	0.115 0.110	0.911 0.917	0.857 0.889	0.963 0.932
OD550(blank-corrected)	0.893 0.867	0.880 0.916	0.890 0.893	0.072 0.076	0.085 0.081	0.071 0.066	0.867 0.873	0.814 0.845	0.919 0.889
mean OD550of the duplicates (blank-corrected)	0.880	0.898	0.892	0.074	0.083	0.068	0.870	0.829	0.904
total mean OD550of 3 replicate tissues (blank corrected)	0.890*			0.075			0.868
SD OD550	0.009			0.007			0.037
relative tissue viabilities [%]	98.9	100.9	100.2	8.3	9.3	7.7	97.7	93.2	101.6
mean relative tissue viability [%]	100.0			8.4**			97.5
SD tissue viability [%]***	1.0			0.8			4.2
CV [% viability]	1.0			9.9			4.3

* Corrected mean OD550 of the negative control corresponds to 100% absolute tissue viability.

** mean relative tissue viability of the three positive control tissues is≤40%

*** The standard deviation (SD) obtained from the three concurrently tested tissues is < 18 %.

3. Quality Criteria

Table 2: Qualitiy Criteria

	Value	Cut off	pass / fail
Mean OD550blank	0.044	< 0.1	pass
Mean Absolute OD550NC	0.934	0.6 ≤ NC ≤ 1.5	pass
Mean Relative Viability [%] PC	8.4	≤ 40 %	pass
SD of Viability [%]	0.8 – 4.2	< 18 %	pass

4. Historical Data

Table 3: Historical Data

	OD550blank	Absolute OD550NC	Relative Viability [%] PC	SD of Viability [%]
Mean	0.043	0.869	12.50	8.01
SD	0.002	0.122	8.67	8.63
n	44	44	44	176

5. Dicussion and Conclusion

The potential of the test item to induce skin irritation was analysed by using the three-dimensional human skin model EPISKIN-SM TM (SkinEthic) comprising a reconstructed epidermis with a functional stratum corneum. In the present study 2’,4’-Bis(methoxycarbonyloxy)-2-brom-acetophenone was applied topically to the EPISKIN-SM TM tissue for 15 min. followed by a 42 h postincubation period and immediate determination of cytotoxic effects via MTT reduction assay. Irritant potential of the test item was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with PBS. The test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 50% (102.3%) after 15 min. treatment and 42 h post incubation. The controls confirmed the validity of the study. The mean OD550 of the six blank values was < 0.1. The mean absolute OD550 of the three negative control tissues was≥ 0.6 and≤ 1.5.The mean relative tissue viability (% negative control) of the positive control was 40 % (8.4 %). The maximum standard deviation of the viability of replicate tissues of all dose groups was < 18 % (0.8 - 4.2 %)

Under the given conditions the test item showed no irritant effects. The test item is therefore classified as “non-irritant” in accordance with UN GHS “No Category”.

Interpretation of results:

GHS criteria not met

Conclusions:

2’,4’-Bis(methoxycarbonyloxy)-2-brom-acetophenone was not irritating in an in vitro skin irritation test (reconstructed human epidermis test method) according to OECD 439 and EU-Method B.46.

Executive summary:

In the present study the skin irritant potential of 2’,4’-Bis(methoxycarbonyloxy)-2-brom-acetophenone was analysed. The EPISKINStandard Model™ (EPISKIN-SMTM), a reconstituted three-dimensional human epidermis model, was used as a replacement for the Draize Skin Irritation Test (OECD TG 404) to distinguish between UN GHS “Category 2” skin irritating test substances and not categorized test substances (“No Category”) which may be considered as non-irritant. Hereby, the test item was applied topically. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 15 min exposure and 42 h post incubation period and compared to those of the concurrent negative controls.

Under the given conditions 2’,4’-Bis(methoxycarbonyloxy)-2-brom-acetophenone showed no irritant effects. The relative mean tissue viability after 15 min of exposure and 42 h post incubation was > 50%. The test item is therefore classified as“non-irritant” in accordance with UN GHS “No Category”.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17.07.2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Controls:

yes, concurrent positive control

yes, concurrent negative control

Duration of treatment / exposure:

240 min

Number of animals or in vitro replicates:

3

Irritation parameter:

in vitro irritation score

Value:

ca. 2.72

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Opacity:

The change of opacity value of each treated cornea or positive and negative control corneae was calculated by subtracting the initial basal opacity from the post treatment opacity reading (t240 – t0), for each individual cornea. The average change in opacity of the negative control corneae was calculated and this value was subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.

Permeability:

The corrected OD490 value of each cornea treated with positive control and test item was calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.

IVIS Calculation:

The following formula was used to determine the IVIS of the negative control: IVIS = opacity value + (15 x OD490 value) The following formula was used to determine the IVIS of the positive control and the test item: IVIS = (opacity value – opacity value mean negative control) + (15 x corrected OD490 value) The mean IVIS value of each treated group was calculated from the IVIS values. Depending on the score obtained, the test item was classified into the following category according to OECD guideline 437:

IVIS	In vitro Irritancy Score (according to OECD 437)
<= 3	No Category (according to GHS)
> 3; <= 55	No prediction can be made
> 55	Serious eye damage according to CLP/EPA/GHS (Cat 1)

The test is acceptable because

• the positive control gives an IVIS that falls within two standard deviations of the current historical mean (updated every three months), and because

• the negative control responses result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control.

Discussion

After a first opacity measurement of the fresh bovine corneae (t0), the 20% (w/v) suspension in saline of the test item 2’,4’-Bis(methoxycarbonyloxy)-2-brom-acetophenone, the positive, and the negative controls were applied to corneae and incubated for 240 minutes at 32 ± 1 °C. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae and opacity was measured again (t240). After the opacity measurements permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C. With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS = 1.04). The positive control (10% (w/v) Benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae (mean IVIS = 128.43) corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)). The mean IVIS of the positive control (mean IVIS = 128.43) was in the range of the historical data (mean IVIS 116.24 ± 2 * 9.31 (see Fig. 3)). Relative to the negative control, the test item 2’,4’-Bis(methoxycarbonyloxy)-2-brom-acetophenone caused an increase of the corneal opacity but not of the permeability. The calculated mean IVIS was 2.72 (threshold for serious eye damage: IVIS ≥ 55). According to OECD 437 the test item is not categorized.

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, according to the current study and under the experimental conditions reported, 2’,4’-Bis(methoxycarbonyloxy)-2-brom-acetophenone is not irritating to eyes.

Executive summary:

An in vitro study was performed to assess the corneal damage potential of 2’,4’-Bis(methoxycarbonyloxy)-2-brom-acetophenone by means of the BCOP assay according to OECD 437 using fresh bovine corneae.

After a first opacity measurement of the fresh bovine corneae (t0), the 20% (w/v) suspension in saline (0.9% (w/v) NaCl in deionised water) of the test item 2’,4’-Bis(methoxycarbonyloxy)-2-brom-acetophenone, the positive, and the negative controls were applied to corneae fixed in an incubation chamber in horizontal position and incubated for 240 minutes at 32 ± 1 °C. The posterior chamber contained incubation medium. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae and opacity was measured again (t240). After the opacity measurements permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C. With the negative control (physiological saline) neither an increase of opacity nor permeability of the corneae could be observed. The positive control (10% (w/v) Benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae corresponding to a classification as serious eye damage (CLP/EPA/GHS (Cat 1)). Relative to the negative control, the test item 2’,4’-Bis(methoxycarbonyloxy)-2-brom-acetophenone an increase of the corneal opacity but not of the permeability compared with the values caused by the negative control. The calculated mean in vitro irritancy score was 2.72. According to OECD 437 the test item is not irritating to eyes.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Under the given conditions 2’,4’-Bis(methoxycarbonyloxy)-2-brom-acetophenone showed no irritant effects in an in vitro skin irritation test (reconstructed human epidermis test according to OECD 439 and EU-Method B.46). The relative mean tissue viability after 15 min of exposure and 42 h post incubation was > 50%. The test item is therefore classified as“non-irritant” in accordance with UN GHS “No Category”.

Furthermore 2',4'-Bis(methoxycarbonyloxy)-2 -brom-acetophenone showed no irritant effects in an in vitro eye irritation test (BCOP-Test according to OECD 437). Relative to the negative control, the test item 2’,4’-Bis(methoxycarbonyloxy)-2-brom-acetophenone caused an increase of the corneal opacity but not of the permeability. The calculated mean IVIS was 2.72. Therefore 2',4'-Bis(methoxycarbonyloxy)-2 -brom-acetophenone does not need to be categorized according to OECD 437.

Justification for classification or non-classification

2',4'-Bis(methoxycarbonyloxy)-2 -brom-acetophenone showed no irritating potential in an in vitro skin irritation test (reconstructed human epidermis test method) according to OECD 439 and EU-Method B.46. Therefore 2',4'-Bis(methoxycarbonyloxy)-2 -brom-acetophenone does not need to be classified as skin irritant.

FFurthermore 2',4'-Bis(methoxycarbonyloxy)-2 -brom-acetophenone showed no irritant effects in an in vitro eye irritation test (BCOP-Test according to OECD 437). Therefore 2',4'-Bis(methoxycarbonyloxy)-2 -brom-acetophenone does not need to be need to be classifed as eye irritant.